Method and system for detection and/or characterization of a biological particle in a sample

ABSTRACT

The present invention provides a method and system for monitoring, detecting, and/or characterizing a biological particle that may be present in a sample whereby the method may be accomplished non-invasively by utilizing a time-dependent spectroscopic technique to obtain at least two measurements of a growth composition comprising a sample and correlating said measurements for the detection and/or characterization of a biological particle, that may be present in the sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is being filed as a divisional of U.S. patentapplication Ser. No. 13/928,857, which was filed Jun. 27, 2013, ispending, and which is a continuation of U.S. patent application Ser. No.12/460,607, which was filed Jul. 22, 2009, and issued as U.S. Pat. No.8,512,975 on Aug. 20, 2013, and claims the benefit of U.S. ProvisionalPatent Application No. 61/135,839, entitled, “Method and System forDetection and/or Characterization of a Biological Particle in a Sample”,filed Jul. 24, 2008.

FIELD OF THE INVENTION

The present invention relates to a method and system for monitoring,detecting and/or characterizing a sample for biological particle growth.

BACKGROUND OF THE INVENTION

Methods to detect and identify microorganisms are typically conducted onseparate automated systems in clinical laboratory and industrialsettings. Standard automated blood culture instruments are restricted toa detection system that gives either a positive or negative result butdoes not provide any information about the characterization or identityof the microorganism. Aside from testing with molecular based assays,which are expensive and generally limited to specific microorganisms,the identity of the microorganism is typically established by taking asample from a positive blood culture bottle and conducting a separateidentification test, usually after an overnight subculture step toprepare the organisms for identification testing.

WO2007/019462 generally relates to a method for the identification andquantification of a biological sample based on measurement of afluorescence Excitation-Emission Matrix (EEM) at a single time point.The sensitivity of this approach is limited because the quenching,fluorescence, and reflectance properties of the surrounding mediumwithin which the biological sample of interest is contained caninterfere with the measurement. If the microorganism is in a homogeneousand transparent sample, such as water or saline, the system is operable.However, for turbid, optically dense, or other complex samples, such asblood, the system is inefficient because the variability of the complexbackground complicates the microorganism spectrum when measured at onlya single time point. Small changes in reflectance and fluorescence ofthe microorganism cannot be measured with a single EEM reading becausethe variability in background signals of turbid samples is greater thanthe specific signal emitted by the microorganism. Thus, sensitivedetection and early characterization or identification of the biologicalentity in a complex sample is generally not practical with a singlemeasurement approach as taught by this reference.

There is a continued need for automated systems to provide additionalcapabilities in monitoring, detecting and/or characterizing biologicalparticles, particularly microorganisms. Alternative methods for earlierdetection and characterization are also desirable because there is abenefit in clinical settings to provide early results to physicians asmore appropriate therapy can be selected at or around the time the bloodor sterile body fluid culture is shown as positive for microbial growth.Additional information in a shortened timeframe would also be helpfulfor non-clinical uses of the system as well.

SUMMARY OF THE INVENTION

Provided herewith is a method and system for monitoring, detectingand/or characterizing biological particles that may be present in asample, useful in both clinical and non-clinical settings.

In one embodiment, the method comprises utilizing a spectroscopictechnique to obtain at least two time-dependent measurements of a growthcomposition comprising a sample and correlating said measurements todetecting and/or characterizing a biological particle if present in saidsample. According to the invention, the measurements take into accountchanges in said growth composition as well as detecting and/orcharacterizing the mass of said biological particle or componentsthereof. The method is particularly useful for monitoring, detectingand/or characterizing microorganisms in complex sample types.

In another embodiment, the invention provides an automated system fordetecting and/or characterizing a biological particle that may bepresent in a sample, said system comprising: (1) a growth chambercomprising a sample and a growth composition; (2) a measurement devicecomprising a reflectance and/or fluorescence spectrometer to enable ameasurement of reflectance and/or fluorescence from said growth chambertaken at two or more time points; and (3) a means for relating saidmeasurement to detecting and/or characterizing said biological particleif present in said sample, wherein said growth chamber is located in thesame system as the measurement device and the measurement isnon-invasive to the growth chamber. In this system, no manual samplingof the composition comprising a sample is required to provide earlydetection and/or characterization, thus providing improved efficiencyand safety that may be particularly useful in both clinical andnon-clinical applications.

In yet another embodiment, a method for detecting and/or characterizinga biological particle present in a sample is provided, said methodcomprising the steps of:

(a) introducing a container comprising a growth composition and a sampleinto a diagnostic system, wherein said container may contain said growthcomposition and said sample either prior to introduction or afterintroduction of said container into said diagnostic system;

(b) illuminating said container at predetermined time points orcontinuously;

(c) monitoring said illuminated composition at predetermined time pointsor continuously to obtain at least two measurements, wherein saidmonitoring is conducted at a wavelength equal to or longer than thewavelength used in illumination, for reflectance and fluorescence,respectively; and

(d) correlating said measurements to detect and/or characterize saidbiological particle if contained within said sample.

In still a further embodiment, a system for detecting and/orcharacterizing biological particle growth is provided, said systemcomprising: (1) a sealed container comprising a sample, a growthcomposition, and a sensor to non-invasively detect growth of saidbiological particle; and (2) a measurement means, such as a spectroscopyapparatus, to provide at least two time-dependent measurements ofreflectance and/or fluorescence of said container in a non-invasivemanner wherein said spectroscopy apparatus provides measurements of bothchanges in sample as well as changes in said growth composition overtime; and (3) a means to correlate said measurements to the detectionand/or characterization of biological particles if present in saidsample. This method is particularly effective in a highly scattering andstrongly fluorescent environment as found in complex sample types suchas blood and other opaque substances. In this system, the combinationoffers diversity within a single system thus providing the test to awider variety of sample types, as discussed in more detail hereinafter.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A and 1B illustrate time-dependent changes in fluorescencesignals of seeded blood cultures containing E. coli and S. aureus,respectably, at several selected wavelengths.

FIGS. 2A and 2B illustrate time-dependent changes in scattering signalsof seeded blood cultures containing E. coli and S. aureus, respectably,monitored at an excitation wavelength of 465 and emission wavelength of465 nm (Ex465/Em465).

FIG. 3 illustrates the percent change in fluorescence signal over timein an E. Coli seeded blood culture with excitation wavelengths of310-320 nm and emission wavelengths of 345-530 nm.

FIGS. 4A and 4B illustrate the Excitation-Emission Matrix (EEM)measurements data obtained from monitoring the fluorescence of an E.coli seeded blood culture, and demonstration of “Early” phase data (FIG.4A) and “Late” phase data (FIG. 4B), respectively.

FIG. 5 illustrates time-dependent change in fluorescence of an E. coliculture in an ethylene oxide (EO) sterilized acrylic cuvette placed inthe 22.5-degree front face waterbath adapter.

FIG. 6 illustrates time-dependent change in fluorescence of an E. coliculture in an EO-sterilized acrylic cuvette placed in the custom-builtautomated carousel adapter.

FIG. 7 illustrates a classification model using general discrminantanalyses of microbial group-specific changes in fluorescence emissionusing seventeen excitation/emission wavelength pairs.

FIGS. 8A-8D illustrate examples of different microorganisms withcharacteristic fluorescence patterns, with FIG. 8A illustrating aNegative Control, FIG. 8B illustrating E. faecalis seeded blood culture,FIG. 8C illustrating P. aeruginosa seeded blood culture, and FIG. 8Dillustrating S. pneumoniae seeded blood culture.

FIGS. 9A-9D illustrate examples of different microorganisms withcharacteristic diffuse reflectance patterns at varying wavelengths, withFIG. 9A illustrating a Negative Control, FIG. 9B illustrating E.faecalis seeded blood culture, FIG. 9C illustrating P. aeruginosa seededblood culture, and FIG. 9D illustrating S. pneumoniae seeded bloodculture.

FIGS. 10A-10F illustrate examples of percent change in fluorescencesignal over time at selected wavelengths for different species ofmicroorganisms.

FIGS. 11A-11D illustrate different classification models using generaldiscrminant analyses of microbial group-specific temporal changes influorescence emission using different numbers of data points in themodel.

FIGS. 12A-12D illustrate examples of different microorganisms withcharacteristic fluorescence patterns grown in commercial blood culturebottles in a proof-of-concept blood culture system.

FIGS. 13A-13D illustrate examples of different microorganisms withcharacteristic fluorescence patterns grown in blood culture bottles in aproof-of-concept blood culture system.

FIG. 14 is a block diagram of a proof-of-concept blood culture system.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a means for detecting relevant levels ofbiological particles and components thereof in a sample. Further,characterization of the biological particles may also be accomplishedbased on observable differences, such as, composition, shape, size,clustering and/or metabolism. The system may be configured to monitorsamples for biological particle growth, detect a biological particle ina sample, characterize a biological particle in a sample, or acombination thereof.

In a continuous analysis configuration, specific signals are determinedby monitoring the fluorescence and reflectance signals of each sample,taken over time and under conditions where the concentration of abiological particle of interest generally increases from a level belowthe detection limit to a detectable level and may be extended to a levelbeyond the detection limit. Using this principle, the system measuressmall changes that may then be correlated with biological particlegrowth and may operate when the sample is of the complex and opaquetype, such as a blood sample in culture media, because the method candistinguish a high background signal from the changes in both the sampleand the growth composition to provide a non-invasive method formonitoring complex sample types. The kinetics of growth are captured inthe method by measuring changes in the sample, changes in the growthcomposition, and/or the actual mass of biological particle.

Samples that may be tested include both clinical and non-clinicalsamples where biological particle presence or growth may be suspected.The amount of sample utilized may vary greatly due to the versatilityand sensitivity of the method. Sample preparation may be accomplished byany number of techniques known to those skilled in the art although oneof the advantages of the present invention is that complex sample types,defined as blood, bodily fluids, or other opaque substances, may betested directly utilizing the system with little or no extensivepretreatment.

Clinical samples that may be tested include any type of sample typicallytested in clinical laboratories, including, but not limited to, blood,sputum, plasma, blood fractions, joint fluid, urine, semen, saliva,feces, cerebrospinal fluid, gastric contents, vaginal secretions, tissuehomogenates, bone marrow aspirates, bone homogenates, sputum, aspirates,swabs and swab rinsates, other body fluids, and the like.

Non-clinical samples that may be tested also include highly variablesubstances, encompassing, but not limited to, foodstuffs, beverages,pharmaceuticals, cosmetics, water, air, soil, plants, blood products(including platelets), donor organ or tissue samples, and the like. Themethod is also particularly well suited for real-time testing to monitorcontamination levels, process control, quality control, and the like inindustrial settings.

As used herein, the terms biological particle and biological particlesare used interchangeably and intended to encompass one or morebiological particle(s) and components thereof that may be detectedand/or characterized in a sample, and includes any organism or cellcapable of self-replication as well as fragments, metabolites, and othersubstances specific to organisms or cells. Encompassed in thisdefinition are microorganisms and non-microorganisms, including, by wayof example, viruses, parasites, protozoans, cryptosporidiums, and thelike, including cell cultures (plant, mammalian, insect, and so on).Particularly well-suited for detection and/or characterization aremicroorganisms, with the term microorganism encompassing organisms thatare generally unicellular, invisible to the naked eye, which can bemultiplied and handled in the laboratory, including but not limited to,Gram-positive or Gram-negative bacteria, yeasts, and molds. By way ofGram-negative bacteria, mention may be made of bacteria of the followinggenera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter,Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella,Vibrio, Yersinia, Acinetobacter, Stenotrophomonas, Brevundimonas,Ralstonia, Achromobacter, Fusobacterium, Prevotella, Branhamella,Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas,Moraxella, Brucella, Pasteurella, Providencia, and Legionella. By way ofGram-positive bacteria, mention may be made of bacteria of the followinggenera: Enterococcus, Streptococcus, Staphylococcus, Bacillus,Paenibacillus, Lactobacillus, Listeria, Peptostreptococcus,Propionibacterium, Clostridium, Bacteroides, Gardnerella, Kocuria,Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria.By way of yeasts and molds, mention may be made of yeasts of thefollowing genera: Candida, Cryptococcus, Nocardia, Penicillium,Alternaria, Rhodotorula, Aspergillus, Fusarium, Saccharomyces andTrichosporon.

According to the invention, there is flexibility in designing the levelof detection and/or characterization that the system may perform.Detection encompasses the observation of at least one change in asample, as determined by at least two, time-dependent measurements,which may be correlated with the presence of a biological particle inthe sample. Detection may occur almost immediately depending upon anumber of factors including the growth rate of the biological particle,fertility of the growth composition, the selectivity of the detectionalgorithm, and/or the time interval of measurement, and so on. Althoughthe actual detection times may vary depending upon these factors,preferred embodiments may provide detection of microorganisms withinabout 48 hours from the initiation of the method, more preferably withinabout 24 hours from the initiation of the method, still more preferablywithin the range of from about 1 hour to about 16 hours from theinitiation of the method, and most preferably within the range of fromabout 1 hour to about 10 hours from the initiation of the method.Characterization encompasses the broad categorization or classificationof biological particles as well as the actual identification of a singlespecies. In some embodiments, classification of the biological particlesof interest may require no prior knowledge of the characteristics of agiven biological particle but only requires consistent correlations withempiric measurements thus making this method more general and readilyadaptable than methods based on specific binding events or metabolicreactions.

More particularly, for microorganism-based applications,characterization may encompass classification of microorganisms into oneor more classification models based on useful information (e.g.,measurements) that may not have been otherwise available within thetimeframe offered by the present method. As used herein, the preferredclassification models comprise grouping or classifying microorganismsinto one or more of the following: (1) Gram Groups; (2) Clinical GramGroups; (3) Therapeutic Groups; (4) Functional Groups; and (5) NaturalIntrinsic Fluorescence Groups.

(1) Gram Groups:

Within the Gram Groups classification, biological particles ormicroorganism may be characterized into one of three broadclassification categories based on their Gram staining reaction andoverall size, said groups may be selected from one or more of the groupsconsisting of: (a) Gram positive microorganisms that stain dark bluewith Gram stain; (b) Gram negative microorganisms that stain red withGram stain; and (c) yeast cells that stain dark blue with Gram stain,but are very large rounded cells that are distinguished from themorphological characteristics of bacteria.

(2) Clinical Gram Groups:

The Gram Groups may be further divided into several sub-categoriesrepresenting distinguishing morphological features. These sub-categoriesmay comprise all the relevant clinical information reported by anexperienced laboratory technologist, and thus provide a higher level ofidentification than a positive or negative Gram reaction. Thisparticular classification may be very helpful because it eliminatesconcerns about relying on the quality of a Gram stain or the skill levelof the technician reading the smear by providing the equivalentclinically relevant information with an automated system. Morespecifically, subcategories of microorganisms based on thisclassification model may be selected from one or more of the groupsconsisting of: (a) Cocci, which are small rounded cells; (b) Diplococci,which are two small rounded cells joined together; (c) Rods, which arerectangular shape; and (d) Bacilli, which are rod shaped. Examples ofadditional morphological information that may be ascertained by thepresent invention include: (i) Gram positive cocci; (ii) Gram positivecocci in chains; (iii) Gram positive cocci in clusters (i.e.,“grape-like” clusters); (iv) Gram positive diplococci; (v) Gram positiverods; (vi) Gram positive rods with endospores; (vii) Gram negative rods;(viii) Gram negative coccobacilli; (ix) Gram negative diplococci; (x)Yeast; and (xi) Filamentous fungi.

(3) Therapeutic Groups:

The therapeutic groups comprise multiple microbial species that, whenisolated from particular specimen types, may be treated with the sameclass of antibiotics or mixture of antibiotics (Reference: “SanfordGuide to Antimicrobial Therapy 2008”). In many cases, identity to thespecies level may not be required by the clinician to enable change frominitial empiric therapy to a more targeted therapy because more than onespecies can be treated with the same choice of antibiotic. Thisclassification level correctly places these “same-treatment”microorganisms into single therapeutic categories. Examples of thischaracterization level include the ability to distinguish highlyresistant enterobacteriacae (EB) species from sensitive EB species(Enterobacter spp. from E. coli), or fluconazole-resistant candidaspecies (C. glabrata and C. kruzei) from sensitive candida species (C.albicans and C. parapsilosis), and so on.

(4) Functional Groups:

According to the invention, microorganisms may also be characterizedinto several groups based upon a mixture of metabolic, virulence andphenotypic characteristics. Non-fermentative organisms may be clearlydistinguished from fermentative ones. Furthermore, microorganism speciesthat produce typical hemolysins may be grouped separately fromnon-hemolytic species. In some cases, these groups may represent broadercategories than genus level (e.g., coliforms, Gram negativenon-fermentative rods), in some cases at the genus level (e.g.,enterococcus, candida), and in some cases closer to species-leveldiscrimination (e.g., coagulase-negative staphylococci, alpha-hemolyticstreptococci, beta-hemolytic streptococci, coagulase-positivestaphylococci, i.e., S. aureus).

(5) Natural Intrinsic Fluorescence (“IF”) Groups:

Microorganisms may also be characterized into categories based on theirinnate or intrinsic fluorescence characteristics. Some of these groupsmay be common to Therapeutic and Functional Group categories. Thesegroupings may be comprise individual species as E. faecalis, S.pyogenes, or P. aeruginosa that have characteristic IF signatures or maycontain small groups of organisms with relatively conserved IFsignatures such as the E. coli-K. oxytoca or E. aerogenes-C. freundiigroups.

According to the invention, the sample is combined with a growthcomposition which is defined as a composition that maintains theviability and/or the growth of the biological particles capable ofself-replicating to be monitored in the system, particularly in a systemthat includes the capability for continuous monitoring. Depending uponthe use of the system, the growth composition may comprise sufficientnutrients to promote rapid growth of the biological particles therebyfacilitating earlier detection and characterization. For example, growthcompositions may comprise media (including agar), liquid culture media(ideal for many uses), or liquid suspension, and the like. Preferably,for microorganism testing, the composition comprises media, such astryptic soy broth, brain heart infusion broth, Columbia broth andBrucella broth, as well as other general purpose complex media known tothose skilled in the art, and may include the addition of bloodsubstitutes and specific growth factors. Additionally, ready-to-usespecially formulated aerobic and anaerobic culture media for thecultivation of a variety of microorganisms may be incorporated into thesystem per the requirements of the organisms of interest. Standard bloodculture media is preferred for more generalized testing, with thefertility of the media and selectivity of the media adjusted as withinthe skill of those familiar with the art. Compositions whereby themicroorganisms grow in a non-homogenous or particulate manner (e.g.Mycobacteria, molds) may also be employed. Adsorbent materials, such asresins, charcoal, Fuller's earth, and the like, may be included in thecomposition to mitigate the effects of samples exposed to antibiotics,as is well known to those skilled in the art.

While the system is adaptable to different growth compositions andsample types, the system is preferably adapted for the variability. Forexample, when the sample comprises blood, algorithms or other means formodeling take into account the background such that the growth of thebiological particle is observable.

In a preferred embodiment, the system includes one or more growthchamber(s) that comprise a temperature-controlled compartment thattypically contains one or more containers comprising a sample and growthcomposition. Optionally, the growth chamber may have an agitationmechanism to provide optimal culture conditions for the growth of thebiological particles, most preferably microorganisms. Appropriate growthconditions are known to those skilled in the art. Both the quantity ofsample and composition in a container may be controlled depending upondesign preferences and the organism of interest. For example, the systemmay include a control component (such as an optical sensor) to regulatethe quantity of sample added to the container in an automated manner.The sample and/or composition may have already been included in thecontainer prior to introduction into the system and samples may includethose that have had growth without continuous monitoring prior tointroduction into the system.

The physical features of the container of the system may take intoaccount the design of the overall system, sample type, growthcomposition, and the like and include optical alignment features tofacilitate optical reading. The container may be constructed from anymaterial that does not interfere with the growth of the biologicalparticle and does not interfere with the measurements taken. Moreparticularly, the container may be constructed from a material that hasgood transmission characteristics in the UV, visible and/or infraredregion of electromagnetic spectrum, with the transmissioncharacteristics present in at least the region where the measurementoccurs. For example, the material may include any UV-VIS-IR transparentmaterial, such as glass or plastic, and ideally will have low gaspermeability. The material may be single or multi-layer, wherepreferably at least one layer has low gas permeability. The containermay be sealed or unsealed and include other desired features within suchas a temperature sensor, carbon dioxide sensor, oxygen sensor,colorimetric sensors, combinations thereof, and the like. For example,the container may have a sensor, preferably an indwelling sensor, tomonitor the temperature of the container that may be electronic oroptical in nature and provide bottle temperature readings whilerequiring no physical contact to the container. The container may be anopened, vented, or closed system. The container may also be designed tofacilitate sampling once adequate microbial growth occurs so that thebiological particles may be isolated and purified for use in an ID, AST,molecular or other diagnostic test system at any time during monitoring.The container may also have an optical surface or coating for collectionof fluorescence and scattered light. Additionally, the container mayincorporate a calibration reference and/or an optical reference indifferent formats, as known to those skilled in the art. For example,the calibration may be accomplished with a feature optically embeddedwith the optical reference present in an inner layer or coating.

In one embodiment, a single system comprising at least two means ofdetecting biological particle growth has been found advantageous becausethe combination of a first and second means of detecting growth in onesystem provides a faster and more specific and sensitive microbialdetection system with a broader applicability to test samples of varyingcomposition. According to the invention, one means of detecting growthin the system comprises the time-dependent measurement of reflectanceand/or fluorescent spectroscopy, as described previously, preferably ina front-face configuration. A second means of detecting growth in thesystem comprises a sealed container comprising a sample, a growthcomposition, and a sensor capable of detecting growth non-invasively.Preferably said sensor is a colorimetric sensor, more preferably aliquid emulsion sensor (LES), as known in the art. However, thetime-dependent reflectance and/or spectroscopy method may provide thepotential for earlier detection of highly metabolically activemicroorganisms, without the need for an internal sensor. Further, themultiple detection indicators (wavelength pairs) may improve systemspecificity while providing for non-invasive detection and/orcharacterization of any biological particles contained in the sample.The second means of a sealed container comprising a sensor may beoptionally included to provide a robust, calibrated internal sensor thatmeasures carbon dioxide and other compounds provided by biologicalparticles. The second means also provides a method of detecting delayedentry positive samples, where the generated signal is not necessarilydependent upon the composition of the test sample but may be derivedfrom the organism itself.

According to the invention, by monitoring the sample in a time-dependentmanner (i.e., by taking at least two time-dependent measurements), thecurrent invention may detect biological particle growth by measuringmultiple changes in the surrounding highly fluorescent environment, andclassify biological particles by continuing to monitor changes untilcharacteristic patterns are recorded and analyzed. More particularly,the method is useful in the area of microorganism detection and/orcharacterization because microorganisms contain, or are composed ofmolecules that fluoresce naturally, depending on specific cellcomposition and metabolism. The resultant patterns differ by organismtype and thus provide a fingerprint per organism type.

Preferably, the system is designed so that the user is automaticallynotified when a biological particle is detected in a sample. Furthercharacterization may be made of the biological particle, as desired.

Once detection occurs, or once the sample has been identified as havingbiological particles present, it has been found that measurabledifferences can form the basis of a method for biological particlecharacterization early in the growth phase, sometimes almostinstantaneous. Characterization patterns may emerge rapidly after theinitial detection, depending upon multiple factors including sampletype, sample concentration, organism concentration in sample, type ofgrowth composition, growth rate of the organism, time interval ofmeasurements, and so on. An automated signal may be provided in thesystem to notify the user upon the characterization of the biologicalparticle, once the biological particle has been characterized into oneor more characterization groups (as described herein) or identified byspecies, etc. In one preferred embodiment, the method automaticallydetects and characterizes microorganisms present within a complex,highly fluorescent and/or optically dense sample, with characterizationspecific to at least the level of information provided by a standardGram stain in the case of bacteria and yeasts. Classificationinformation may be extracted at any point during the growth of thebiological particle once the sample is introduced into the system.

Preferably characterization occurs within about 48 hours of initialdetection, more preferably within about 24 hours of initial detection,still more preferably within about 0 to about 16 hours of initialdetection, and most preferably within from about 0 to about 8 hours postinitial detection. For example, characterization may begin to occur in“early phase” (change occurring from about 0 to about 2 hours postinitial detection or positive growth signal) and/or “late phase” (changeoccurring from about 2 to about 8 hours post initial detection orpositive growth signal). Early phase tends to show patterns wherespectra are dominated by changes in the growth composition. Late phasetypically shows patterns where spectra are dominated by the biologicalparticle mass rather than by changes in the growth composition.

The length of time in which monitoring the sample may occur may varywidely according to the needs of the user. For example, testing mayoccur for a period of time between testing initiation to days or evenmonths, depending upon the biological particle of interest, etc., asknown to those skilled in the art.

When monitoring for biological particle growth, preferably formicroorganism growth, the sample may be excited as frequently as theuser finds helpful for the particular testing needs, and may beautomated by software. For example, for typical microorganism monitoringin blood or body fluid testing, the sample may be excited constantly orperiodically. More particularly, the frequency of exciting the samplemay be adjusted anywhere from constant excitation to excitation everyfew hours or every few days or so, more preferably within the range ofexciting the sample every minute or so to every three hours, and mostpreferably within the range of from about every five minutes to aboutevery hour. As used herein, excitation and illumination are usedinterchangeably.

Growth of the self-replicating biological particles may be monitored inreal-time within a growth chamber where the readings are conductedwithout requiring the removal of the sample by selection of a systemthat is automated. While continuous monitoring is of particularusefulness, the method may be alternatively configured to monitor,detect and/or characterize biological particles present in a sample byperiodic scanning, random access, and the like.

The sample illumination source, or excitation source, may be selectedfrom any number of suitable light sources as known to those skilled inthe art. More preferably, light sources capable of emission in theultraviolet, visible and near-infrared portions of the electromagneticspectrum are utilized and are known to those skilled in the art. Forexample, light sources may be continuum lamps such as a deuterium orxenon arc lamp for generation of ultraviolet light and a tungstenhalogen lamp for generation of visible/near-infrared excitation. Theselight sources provide a broad range of emission, and the spectralbandwidth for specific excitation wavelengths may be reduced usingoptical interference filters, prisms or optical gratings.

Alternatively, a plurality of narrowband light sources, such as lightemitting diodes or lasers, may be spatially multiplexed to provide amulti-wavelength excitation source. For example, currently, lightemitting diodes are available from 240 nm to in excess of 900 nm and thesources have a spectral bandwidth of 20-40 nm (full width at halfmaximum). Lasers are available in discrete wavelengths from theultraviolet to the near-infrared; many multiplexing methods are known tothose skilled in the art.

The spectral selectivity of any of the light sources may be improved byusing spectral discrimination means such as a scanning monochromator.Other methods of discrimination may be utilized by persons skilled inthe art such as an acousto-optic tunable filter, liquid crystal tunablefilter, an array of optical interference filters, prism spectrograph,etc. A consideration in selecting the spectral discriminator takes intoaccount the range of tunability as well as the level of selectivity. Byway of illustration, for example, a discriminator might utilize thewavelength range of 300-800 nm with a selectivity of 10 nm. Theseparameters generally determine the optimum technology necessary toachieve the tunability range as well as the selectivity.

Typically, the light source results in the excitation of the samplefollowed by measurement of the emission of fluorescence of the sample atpredetermined time points or continuously. Similarly, the reflectedlight (i.e., scattered light) from the excitation source's interactionwith the sample may be measured and has been shown to provide pertinentdata for detection and characterization.

The emission from the sample may be measured by any suitable means ofspectral discrimination, most preferably employing a spectrometer. Thespectrometer may be a scanning monochromator that detects specificemission wavelengths whereby the output from the monochromator isdetected by a photomultiplier tube or the spectrometer may be configuredas an imaging spectrograph whereby the output is detected by an imagingdetector array such as a charge-coupled device (CCD) detector array.Other methods of discrimination may be utilized by persons skilled inthe art such as an acousto-optic tunable filter, liquid crystal tunablefilter, an array of optical interference filters, prism spectrograph,etc. In a preferred embodiment, a discriminator allows the observationof the fluorescence and/or scattering signal by a photodetection means(such as a photomultiplier tube, avalanche photodiode, charge coupleddevice (CCD) detector array, or electron multiplying charge coupleddevice (EMCCD) detector array).

The time-dependent spectroscopic technique is used to obtain at leasttwo measurements that are preferably provided as Excitation-EmissionMatrix (EEM) measurements. As used herein, EEM is defined as theluminescent spectral emission intensity of fluorescent substances as afunction of both excitation and emission wavelength, and may include afull spectrum or a subset thereof, wherein a subset may contain a singleor multiple excitation/emission pairs(s). FIGS. 4A and B show contourplots of time-dependent changes over the entire EEM spectra.Additionally, a cross section of the EEM with a fixed excitationwavelength may be used to show the emission spectra for a specificexcitation wavelength, and a cross section of the EEM with a fixedemission wavelength may be used to show the excitation spectra for asample. In one embodiment, multiple EEMs are measured at discrete pointsin time and using specific excitation-emission wavelength pairs.

In accordance with one embodiment, it has been found that front-facefluorescence spectroscopy provides an advantage in measuring thefluorescence and reflectance properties of highly scattering and highlyquenching samples. The front-face method is particularly usefulspectroscopic method because it has been found that this configurationis less affected by the interfering components of blood andmicrobiological culture media. In accordance with this embodiment, theoptical surface of the container may be illuminated at such an angle asto provide acceptable results as known to those skilled in the art,(e.g., Eisinger, J., and J. Flores, 1983, “Front-face fluorometry ofliquid samples,” Anal. Biochem. 94:15-21). More particularly, theillumination may occur at any angle wherein the specular refection isnot directed into the detector. Preferably, the system is designed suchthat the spectroscopy measures diffuse reflected light at a minimum ofone fixed angle in addition to measuring emitted fluorescence at aminimum of one fixed angle. By way of example, the optical surface ofthe container may be positioned in the front-face configuration andilluminated at an angle of 0 to 90 degrees normal to the surface of thecontainer.

According to the invention, control measurements (e.g., fluorescenceand/or reflectance measurements) are taken for known biologicalparticles (preferably microorganisms) in specific sample types thusallowing for correlation of measured test data with characterization ofthe biological particles of interest using various mathematical methodsknown to those skilled in the art. For example, the data from samplesmay be compared with the baseline or control measurements utilizingsoftware systems known to one skilled in the art. More particularly, thefluorescence and scattering data may be analyzed by a number ofmultivariate analysis methods, such as, for example, GeneralDiscriminant Analysis (GDA), Partial Least Squares Discriminant Analysis(PLSDA), Partial Least Squares regression, Principal Component Analysis(PCA), Parallel Factor Analysis (PARAFAC), Neural Network Analysis (NNA)and Support Vector Machine (SVM). These methods may be used to classifyunknown biological particles of interest (preferably a select group ofmicroorganisms) into relevant groups based on existing nomenclature, orinto naturally occurring groups based on the organism's metabolism,pathogenicity and virulence in designing the system for monitoring,detecting and/or characterizing the organism as described previously.

In a preferred embodiment, the system detects microorganisms usingchange in intrinsic fluorescence and/or reflectance of the culture mediaand sample, taking into account the growth of the microorganism itselfand the changes in the kinetics due to metabolism of the microorganismin the culture. The intrinsic fluorescence or auto fluorescence of themicroorganism, particularly bacteria, leverages the fact that thebacteria contain natural fluorophores such as aromatic amino acids(e.g., tryptophan, tyrosine, phenylalanine) that can be excited via amulti-wavelength light source.

The container used in the system to hold the sample may further comprisea combined CO₂ or other sensor that may be included for any number ofreasons, including, but not limited to, compatibility with previoussystems; contamination detection during manufacturing, transport orstorage; and accommodation of delayed entry of bottles into anincubation/reading system. Still further, the container may haveincluded a radio frequency identification device, barcode, or the liketo store data from an initial read of the bottle at time of samplecollection (including time), information from a test (could be used forpost characterization), manufacturing information (lot, date,expiration, initial readings, etc.), patient and sample information attime acquired at the time of collecting the sample, and the like.

The present invention is further detailed in the following examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized.

EXAMPLES

A Fluorolog 3 fluorescence spectrophotometer system (Horiba Jobin-Yvon)was modified with a temperature-controlled front-face cuvette holder toenable the incubation and continuous monitoring of seeded blood culturescontained in sterile cuvettes. The fluorescence was recorded using aphotomultiplier tube. Fluorescence signal strength was taken at severaldifferent wavelengths and saved. The readings were compared toexperimentally determined strengths for different types ofmicroorganism. In addition, further analysis of the spectra wasconducted so that microbial identification to a given level ofconfidence was achieved.

Example 1 describes seeded blood cultures in autoclaved quartz cuvettes.Ethylene-oxide sterilized acrylic cuvettes were used in Example 2. Acustom-built carousel adapter that incubated the cultures at 35-37° C.and enabled multiple cultures to be performed simultaneously was used inExample 3. These experiments demonstrated that the invention possessesthe potential ability to detect microorganisms in blood culture earlierthan the current state-of-the art CO₂ sensors and has capabilities toidentify the isolate at least to a clinically useful classificationlevel. A database of EEM and scattering data from blood cultures ofapproximately 80 microorganism strains was built. Several multivariateanalysis methods were used to classify unknown strains. The results ofthe General Discriminant Analysis (GDA) method are presented in Example4. Optimization of the front-face angle for collection of blood cultureEEM and scattering spectra is described in Example 5. Non-invasiveidentification of microorganisms growing in seeded blood cultures isdescribed in Example 6. Example 7 describes the results obtained with aProof-of-Principle (POP) blood culture system using existing commercialblood culture bottles and continuous monitoring with multiplefluorescence and reflectance wavelengths.

Example 1: Blood Culture (Culture Medium and Blood Sample) in QuartzCuvette in Waterbath Adapter

Seeded blood cultures were set up in autoclaved 1.0 cm screw-cappedquartz cuvettes (Starna, Inc.) containing a stir bar for agitation. Tothe cuvette was added 2.4 mL of standard blood culture medium, 0.6 mL offresh normal human blood and 0.05 mL of a 10³/mL suspension of testmicroorganism (approx. 10 CFU/cuvette). A sterile, septum screw cap wasplaced on the cuvette, and it was inserted into the front-face adapterpreviously described. The culture was maintained at approximately 36° C.by connecting the adapter to a recirculating water bath heated to 36° C.The cuvette was read every 45 minutes by the Fluorolog 3 fluorescencespectrophotometer that was software controlled. A full EEM spectra wascollected at each time point with an Excitation wavelength range of260-580 nm (every 5 nm) and an Emission wavelength range of 260-680 nm(every 5 nm) for a total of 3,139 data-points per scan. Each scan tookapproximately 23 minutes to complete. The cultures were maintained, andmeasurements taken continuously, for up to 24 hours.

Examples of the changes in fluorescence signal of severalExcitation-Emission wavelength pairs for E. coli and S. aureus culturesare shown in FIGS. 1A and 1B. It is clear that following the initialpoint of detection, the temporal changes occurring at these wavelengthsare significantly different between the two organisms. Examples of thechanges in diffuse reflectance signal at 465-465 nm for E. coli and S.aureus cultures are shown in FIGS. 2A and 2B. A clear difference in theshape of the curves over time was observed.

Further detailed examination of the changes in fluorescence from all3,139 data-points of an E. coli culture revealed the presence of atleast two visually-identifiable phases; change from 7-10 hrs(approximately 0-2 hours after initial detection) of culture primarilycomprising the rapid initial change in the fluorescence of the culturemedium, and a change from 10-15 hr (approximately 2-7 hours afterinitial detection) that reflects an increase of microbial intrinsicfluorophores. This phenomenon is shown in FIG. 3 as a line plot ofExcitation wavelengths from about 310-320 nm and Emission wavelengthsfrom about 345-530 nm, and in FIGS. 4A and 4B as contour plots oftime-dependent changes over the entire EEM spectra. FIGS. 3, 4A and 4Bdemonstrate “early” and “late” phase changes that can be used for thedetection and/or characterization of a biological particle. This dataexemplifies the power of temporal fluorescence and scatteringmeasurements of a growing microbial culture.

Example 2: Blood Culture in Acrylic Cuvette in Waterbath Adapter

An E. coli blood culture was set up as described in Example 1 with theexception that the cuvette was constructed of a UV-transparent acrylic(Sarstedt 67.755), and sterilized by ethylene oxide treatment. Thechange in the autofluorescence or intrinsic fluorescence of theblood-media mixture over time with multiple wavelength pairs is shown inFIG. 5.

Example 3: Blood Culture in Acrylic Cuvette in Multi-Station CarouselAdapter

An E. coli blood culture was set up as described in Example 2 with theexception that the cuvette was loaded into a custom-built,temperature-controlled carousel adapter for the Fluorolog 3 system. Thechange in the autofluorescence or intrinsic fluorescence of theblood-media mixture over time with multiple wavelength pairs is shown inFIG. 6.

The experiments described in Examples 2 and 3 demonstrate thattime-dependent changes in fluorescence were measured in readilyavailable plastic containers and in a manner amenable to larger scaleautomation, respectively. The same is true for the diffuse reflectancemeasurements collected in these experiments (data not shown).

Example 4: GDA Analysis of Simulated Blood Culture Study (Group Level)

In this study, a sample of positive culture medium was removed fromseeded BacT/ALERT® SA (bioMérieux, Inc.) blood cultures bottles within afew minutes of the BacT/ALERT® Microbial Detection System (bioMérieux,Inc.) calling the culture positive. Culture media removed from sterileblood culture bottles at similar times served as negative controls. Theculture media samples were placed in acrylic cuvettes and read in theFluorolog 3 with a full EEM scan. Fluorescence and scattering data werenormalized to age-matched negative controls, and then analyzed byGeneral Discriminant Analysis (GDA).

Measurements taken for multiple data points from each sample tested werecompared to a database of EEM and reflectance data from blood culturesof 77 known microorganism strains, representing 12 species, and thetested strains were classified based on the comparisons. The percentageof strains correctly identified to the “Group” classification levelbased on the number of data points collected is presented in FIG. 7.FIG. 7 demonstrates that analysis of microbial-specific changes influorescence and scattering classified >97% of the test microorganismscorrectly into clinically relevant groups based on either existingnomenclature or the organism's innate metabolism, pathogenicity andvirulence characteristics.

Example 5: Optimization of the Front-Face Angle

Blood cultures of E. coli in acrylic cuvettes were set up as describedin Example 3. The front face angle of sequential cultures was adjustedto test the following angles: 20, 22.5, 26, 30, 34 and 38 degrees. Puresuspensions of E. coli and S. aureus were also measured at each angle.The optimal front face angle was shown to be 26 degrees, when assessedas either the greatest degree of change in the E. coli cultures (Table1), or as the highest absolute signals and signal to noise ratios ofcellular fluorophores of the two microbial suspensions (data not shown).The values given in Table 1 represent the average signal for allRayleigh points (260-750 nm) and all fluorescence points within the EEM.While the average Rayleigh signal in positive regions of the microbialgrowth curve was reduced by 66%, large increases were observed atspecific wavelengths such as 465-465 nm, as exemplified in FIGS. 2A and2B.

TABLE 1 % Change in Signal from Negative to Positive Regions of an E.coli Growth Curve Front-Face Angle 20° 22.5° 26° 30° 34° 38° Scattering−50% −59% −66% −49% −43% −62% Average EEM 1% 1% 10% 2% 0% −2% Average

Example 6: Non-Invasive Identification of Microorganisms Growing inBlood Cultures

A total of 119 seeded blood cultures, representing 5-15 strains offourteen clinically-relevant species (see Table 2 below), wereinoculated into sterile acrylic cuvettes and loaded into themulti-station carousel adapter for the FluoroLog 3 system.

TABLE 2 Clinically Relevant Species Tested C. albicans E. coli A.baumanii E. faecalis C. parapsilosis E. aerogenes P. aeruginosa S.pneumoniae C. tropicalis K. pneumoniae S. aureus S. mitis P. mirabilisS. epidermidisEach culture was scanned continuously (every 30 to 35 minutes) for up to5 days using a selection of 82 fluorescent wavelength pairs and 50diffuse scattering wavelengths.

FIGS. 8B-8D show characteristic fluorescence patterns over a period of23 hours for different microorganisms. The data from 4 of the 82wavelength pairs are shown (Ex360/Em380 nm, Ex460/Em480 nm, Ex650/Em670nm and Ex700/Em730 nm). FIG. 8A shows data collected for a NegativeControl, FIG. 8B shows data collected for a E. faecalis seeded bloodculture, FIG. 8C shows data collected for a P. aeruginosa seeded bloodculture, and FIG. 8D shows data collected for a S. pneumoniae seededblood culture. Obvious differences in the shapes of the fluorescentcurves are evident between these three species.

FIGS. 9B-9D show characteristic diffuse reflectance patterns atwavelengths from 280-720 nm over a 23-hour culture period for the samethree microorganisms shown in FIG. 8. FIG. 9A shows data collected for aNegative Control, FIG. 9B shows data collected for E. faecalis, FIG. 9Cshows data collected for P. aeruginosa, and FIG. 9D shows data collectedfor S. pneumoniae. Once again, distinctive patterns of reflectanceemerged, as indicated by a shift away from the normal reflectance signalof the negative control (FIG. 9A). The extent and rate of this temporalshift in reflectance was characteristic for certain microorganisms.

FIGS. 10A-10F shows the percent change in fluorescence signal over timeat six selected wavelengths for eleven species of microorganisms. FIG.10A shows data collected with an excitation wavelength of 300 nm and anemission wavelength of 490 nm for various species, FIG. 10B shows datacollected with an excitation wavelength of 360 nm and an emissionwavelength of 380 nm for various species, FIG. 10C shows data collectedwith an excitation wavelength of 460 nm and an emission wavelength of480 nm for various species, FIG. 10D shows data collected with anexcitation wavelength of 480 nm and an emission wavelength of 510 nm forvarious species, FIG. 10E shows data collected with an excitationwavelength of 520 nm and an emission wavelength of 740 nm for variousspecies, and FIG. 10F shows data collected with an excitation wavelengthof 610 nm and an emission wavelength of 640 nm for various species.

The measurements and data collected for characteristic fluorescenceand/or diffuse reflectance patterns over time, and/or change influorescence signal over time at multiple wavelengths can be compared toa database of EEM and scattering data from known microorganism strains,and used to detect and characterize a biological particle that may bepresent in a sample.

FIGS. 11A-11D show the percentage of strains correctly identified todifferent classification models using general discriminant analyses ofmicrobial group-specific temporal changes in fluorescence and/or diffusereflectance data using a different numbers of data points in the model.Measurements taken for multiple data points from each sample tested werecompared to a database of EEM and reflectance data from blood culturesof approximately 120 known microorganism strains, and each test strainwas classified based on the comparisons. FIG. 11A shows classificationby Clinical Gram group, and shows that approximately 93% of speciestested were correctly identified to the Clinical Gram level 0-4 hoursafter detection based on 11 data points. FIG. 11B shows classificationby Therapeutic Group level, and shows that approximately 97% of speciestested were correctly characterized 0-4 hours after detection based onthe same 11 data points. FIG. 11C shows classification by TherapeuticGroup level, and shows that approximately 96% of species tested werecorrectly characterized 0-7 hours after detection based on the same 11data points. FIG. 11D shows classification by Therapeutic Group, andshows that approximately 98% of species tested were correctlycharacterized 0-7 hours after detection based on a different input of 21data points.

FIGS. 11A-11D demonstrate that analysis of microbial-specific changes influorescence can be used to classify microorganisms into clinicallyrelevant groups or categories based on either existing nomenclature orthe organism's innate metabolism, pathogenicity and virulencecharacteristics.

Example 7: Proof-of-Concept Blood Culture System Using Commercial BloodCulture Bottles

A proof-of-concept blood culture system was developed to demonstrate thecapabilities of the new technology in commercially manufacturedBacT/ALERT® (bioMérieux, Inc.) culture bottles with and without theindwelling Liquid Emulsion Sensor (LES). This system was capable oftesting one bottle at a time using the same temperature and agitationconditions as the commercial BacT/ALERT® Microbial Detection System(bioMérieux, Inc.).

Two light sources were used to generate the excitation light; one was a470 nm laser and the other was a 650 nm laser. Coherent light sourceswere used so that an evaluation could be done with narrow Stokes shifts.It is also advantageous to use a coherent light source in a fiber opticapplication since the optical coupling efficiency is higher with alaser. Narrowband optical filters restrict fluorescence (or amplifiedspontaneous emission) from entering into the fiber; this ensures thefluorescence signal alone is from fluorophores of interest in thesample. The filtered light from each of the sources is coupled into thebifurcated fiber using fiber optic coupling optics (well known to thosein the field). The birfurcated fiber combines the light of two fibersinto one which is incident onto the sample bottle. A classicalreflectance probe configuration (6 collection fibers around 1 excitationfiber was used). The collection fibers collect the reflected excitationlight and the fluorescent light and couple it back to a bifurcatingfiber (which splits the light into two paths). The light is thenextracted from the fiber optics and collimated using a collimationoptical assembly. Bandpass filters are used to separate the fluorescencefrom each channel so that one photodetection system detects thefluorescence from the 470 nm source and the other detects thefluorescence from the 650 nm source. Each of the filters blocks theexcitation light frequency. A block diagram of the system is shown inFIG. 14.

Plastic BacT/ALERT® SA (bioMérieux, Inc.) culture bottles were filledwith 10 mL of normal human blood or defibrinated horse blood and seededwith 10²-10³ CFU of a variety of microorganisms. Bottles were loadedinto the instrument and read every 10 minutes for up to 120 hours. Thefiber optic probe was placed adjacent to the side-wall of the rockingculture bottle at angles between 0 and 90 degrees. Additionally,BacT/ALERT® SA bottles were made without the indwelling Liquid EmulsionSensor (LES) and the fiber optic probe placed perpendicular to thebottom of the tube to collect data.

The results of several side-wall read cultures are shown in FIGS.12A-12D for S. pneumoniae, E. faecalis, P. mirabilis and C. hominisrespectively. Obvious changes in fluorescence intensity occurred due tomicrobial growth for both test wavelengths (Ex650/Em670 nm andEx470/Em670 nm). Furthermore, the extent, rate and direction of thefluorescence signal differed between the species tested.

The results of several bottom-wall read cultures are shown in FIGS.13A-13D for S. pneumoniae, P. mirabilis, S. aureus and E. colirespectively in BacT/ALERT® SA bottles made without the indwellingLiquid Emulsion Sensor (LES). Characteristic changes in fluorescenceintensity, rate and direction were observed for these species ofmicroorganisms within a few hours of initial detection.

FIGS. 12A-D and 13A-D demonstrate that analysis of microbial-specificchanges in fluorescence can be used to classify microorganisms intoclinically relevant groups or categories based on either existingnomenclature or the organism's innate metabolism, pathogenicity andvirulence characteristics.

That which is claimed is:
 1. An automated system for characterizing amicroorganism that is present in a blood sample, said system comprising:(1) a growth chamber comprising a blood culture medium and said bloodsample, a blood culture being formed on combining said blood sample withsaid blood culture medium; (2) a measurement device comprising atime-dependent reflectance and fluorescence spectrometer to obtainreflectance and intrinsic fluorescence measurements from said bloodculture at two or more time points, wherein said at least twomeasurements obtained from said blood culture at two or more time pointscomprise directly measured reflectance and intrinsic fluorescencemeasurements of said blood culture from said measurement device; and (3)computer software configured to correlate said measurements to identifysaid microorganism to species, wherein said growth chamber is locatedwithin said spectrometer and said measurement is non-invasive to saidgrowth chamber.
 2. The automated system of claim 1 wherein saidcorrelation comprises a comparison of time-dependent reflectance andintrinsic fluorescence measurements at said two or more time points withcontrol measurements taken for known microorganisms.
 3. The automatedsystem of claim 2 wherein said comparison is done using software.
 4. Thesystem according to claim 1 wherein said spectrometer is configured tomonitor growth parameters of microorganisms and changes in complexculture media due to metabolic activity of the microorganisms.
 5. Thesystem according to claim 1 wherein said growth chamber is a sealedcontainer.
 6. The system according to claim 1 wherein said growthchamber further comprises a sensor to non-invasively detect growth ofmicroorganism.
 7. The system according to claim 1 further comprising anautomated notification device that provides a signal upon detection ofsaid microorganism.
 8. The system according to claim 7 wherein thesystem further comprises a second automated notification device thatprovides a signal upon characterization of said microorganism.
 9. Amethod for characterizing a microorganism in a blood sample, said methodcomprising: (a) introducing a container comprising said blood sample anda blood culture medium into the automated system for characterizing amicroorganism as claimed in claim 1, wherein said blood sample and bloodculture medium can be included in said container prior to introductionor after introduction of said container into said automated system forcharacterizing a microorganism; (b) illuminating said container at oneor more predetermined time points or continuously; (c) monitoring saidilluminated composition at said one or more predetermined time points orcontinuously to obtain at least two measurements, wherein saidmonitoring is conducted at a wavelength equal to or longer than thewavelength used in illumination, for reflectance and fluorescence,respectively; and (d) correlating said measurements to identify saidmicroorganism to species.
 10. The method according to claim 9 whereinsaid correlation comprises a comparison of said at least twomeasurements with control measurements taken for known microorganisms.